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Functional characterization of the promoter for the mouse SPTLC2 gene, which encodes subunit 2 of serine palmitoyltransferase.

ABSTRACT: A series of luciferase reporter constructs was prepared from a 1035-bp fragment of mouse genomic DNA flanking the 5'-coding sequence for the SPTLC2 subunit of serine palmitoyltransferase, the initial enzyme of de novo sphingolipid biosynthesis. The full-length DNA fragment promoted strong reporter gene expression in NIH3T3 cells while deletion and site-directed mutagenesis indicated that the proximal 335 bp contain initiator and downstream promoter elements, two proximal GC boxes that appear to stimulate transcription in a cooperative manner, and several additional elements whose activity cannot be accounted for by known factor binding sites. These findings provide insight into the control mechanisms for transcription of mammalian SPTLC2.

SUBMITTER: Linn SC

PROVIDER: S-EPMC1698862 | biostudies-literature | 2006 Nov

REPOSITORIES: biostudies-literature

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Functional characterization of the promoter for the mouse SPTLC2 gene, which encodes subunit 2 of serine palmitoyltransferase.

Linn Stephen C SC Andras Lindsay M LM Kim Hee-Sook HS Wei Jia J Nagiec M Marek MM Dickson Robert C RC Merrill Alfred H AH

FEBS letters 20061019 26

A series of luciferase reporter constructs was prepared from a 1035-bp fragment of mouse genomic DNA flanking the 5'-coding sequence for the SPTLC2 subunit of serine palmitoyltransferase, the initial enzyme of de novo sphingolipid biosynthesis. The full-length DNA fragment promoted strong reporter gene expression in NIH3T3 cells while deletion and site-directed mutagenesis indicated that the proximal 335 bp contain initiator and downstream promoter elements, two proximal GC boxes that appear to ...[more]

PMID: 17070807

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Project description:BACKGROUND:Glutaminase is expressed in most mammalian tissues and cancer cells, but the regulation of its expression is poorly understood. An essential step to accomplish this goal is the characterization of its species- and cell-specific isoenzyme pattern of expression. Our aim was to identify and characterize transcript variants of the mammalian glutaminase Gls2 gene. METHODOLOGY/PRINCIPAL FINDINGS:We demonstrate for the first time simultaneous expression of two transcript variants from the Gls2 gene in human, rat and mouse. A combination of RT-PCR, primer-extension analysis, bioinformatics, real-time PCR, in vitro transcription and translation and immunoblot analysis was applied to investigate GLS2 transcripts in mammalian tissues. Short (LGA) and long (GAB) transcript forms were isolated in brain and liver tissue of human, rat and mouse. The short LGA transcript arises by a combination of two mechanisms of transcriptional modulation: alternative transcription initiation and alternative promoter. The LGA variant contains both the transcription start site (TSS) and the alternative promoter in the first intron of the Gls2 gene. The full human LGA transcript has two in-frame ATGs in the first exon, which are missing in orthologous rat and mouse transcripts. In vitro transcription and translation of human LGA yielded two polypeptides of the predicted size, but only the canonical full-length protein displayed catalytic activity. Relative abundance of GAB and LGA transcripts showed marked variations depending on species and tissues analyzed. CONCLUSIONS/SIGNIFICANCE:This is the first report demonstrating expression of alternative transcripts of the mammalian Gls2 gene. Transcriptional mechanisms giving rise to GLS2 variants and isolation of novel GLS2 transcripts in human, rat and mouse are presented. Results were also confirmed at the protein level, where catalytic activity was demonstrated for the human LGA protein. Relative abundance of GAB and LGA transcripts was species- and tissue-specific providing evidence of a differential regulation of GLS2 transcripts in mammals.

Dataset Information

Functional characterization of the promoter for the mouse SPTLC2 gene, which encodes subunit 2 of serine palmitoyltransferase.

Publications

Functional characterization of the promoter for the mouse SPTLC2 gene, which encodes subunit 2 of serine palmitoyltransferase.

Similar Datasets

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